Capillary electrophoresis and capillary chromatography provide a number of distinct advantages as separation processes for biochemical mixtures. One advantage is the small volume of the capillary interior. This permits one to perform separations on extremely small samples, and at high speed. Another advantage, which pertains specifically to electrophoresis in capillaries, is the rapid rate at which heat is dissipated outward from a capillary due to the capillary's narrow bore. This permits the use of a high voltage to drive the electrophoresis, which provides separations at high speed and efficiency. Each of these advantages renders capillaries particularly useful for analyzing samples of biological interest, particularly mixtures of peptides, proteins and nucleic acids.
The most advantageous method of detecting separated solutes in capillary separations is on-line detection. One of several advantages which on-line detection offers is the capability of obtaining a multitude of chromatogram and electropherogram traces during the course of a single continuous electrophoretic separation. The most common method of achieving this is by scanning the capillary several times while the separation is in progress. This permits the operator to follow the course of a separation, to determine solute mobilities and peak areas on the basis of several measurements rather than just one, and to select a chromatogram and an electropherogram which has the greatest peak separation with the least broadening.
One difficulty with scanning is the need for either a moving detector or a moving capillary, and the need to accurately correlate the relative positions of the detector and capillary with the peaks observed. Another difficulty is the need to use a capillary made of quartz which is ground and polished both inside and outside to minimize variations in internal diameter and wall thickness which might otherwise produce baseline fluctuations. A third difficulty is the need for both the capillary and the medium inside the capillary to be transparent to a detection beam such as ultraviolet (UV) light, along the entire length of the capillary along which scanning will be performed. In many cases, coatings are applied to the outer walls of capillaries for purposes of rendering the capillary more flexible and less prone to breakage. Many of these coatings are opaque to UV light, and thus do not permit scanning.
The use of two detectors mounted at a fixed distance from each other is disclosed by Beckers, J.L., et al., "Use of a Double-Detector System for the Measurement of Mobilities in Zone Electrophoresis," J. Chromatog. 452: 591-600 (1988). Such a system is less than fully satisfactory, however. The equipment cost for a two-detector system of course includes the cost of two detectors, which are a major component of the cost of the system as a whole. Also, two detectors seldom have the same sensitivity, and differences in sensitivity will result in different peak areas for a particular solute. Still further, the use of two detectors imposes restrictions on how the detectors may be positioned relative to each other, i.e., there is a limit to how closely they can be placed unless they utilize fiber optics.
These and other problems are addressed by the present invention.